The goal of this proposal is to elucidate the molecular and morphological mechanisms leading to the differentiation of the definitive endoderm. The definitive endoderm, one of the three primary germ layers produced during gastrulation, emerges as an epithelial sheet that lines the entire ventral surface of the presomitic mouse embryo. The cells of this epithelial sheet become internalized and form the digestive tract and associated digestive organs, a process that involves inductive interactions with the underlying mesoderm. Although many studies have focused on the endoderm after the onset of organogenesis, very little is known about the endoderm prior to this stage of differentiation. This proposal will elucidate the initial processes leading to organogenesis by exploiting the accessibility of the early definitive endoderm in a whole embryo culture system. First, a fate map of the early endoderm will be produced by manually labeling groups or individual cells in the epithelial sheet with a fluorescent dye, culturing the embryos through organogenesis and determining the contribution of the descendants by whole mount and section fluorescence. A second set of experiments will be performed to understand the inductive tissue interactions involved in organogenesis. Specifically, histological and fate mapping techniques will be used to study the dynamic mesodermal interactions that physically underlie and thus potentially influence the pre-liver and pancreas domains. Finally, the exposure of the early endoderm in the embryo culture system will be exploited by using gene knockdown and signal pathway inhibitors as novel approaches to assessing gene redundancy and initial screens of function. Together these studies will provide for a more thorough understanding of the processes leading to the formation of a normal digestive system, allowing for the advancement of therapies for the diseased state.